Simultaneous Genotyping of the rs4762 and rs699 Polymorphisms in Angiotensinogen Gene and Correlation with Iranian CAD Patients with Novel Hexa-primer ARMS-PCR.

BACKGROUND
A significant role of Renin-angiotensin system (RAS) genetic variants in the pathogenesis of essential hypertension and cardiovascular diseases has been proved. This study aimed to develop a new, fast and cheap method for the simultaneous detection of two missense single nucleotide polymorphisms (T207M or rs4762 and M268T orrs699) of angiotensinogen (AGT) in single-step Multiplex Hexa-Primer Amplification Refractory Mutation System - polymerase chain reaction (H-ARMS-PCR).


METHODS
In this case-control study, 148 patients with coronary artery disease (CAD) and 135 controls were included. The patients were referred to cardiac centers in Afshar Hospital (Yazd, Iran) from 2012 to 2015. Two sets of inner primer (for each SNP) and one set outer primer pairs were designed for genotyping of rs4762 and rs699 in single tube H-ARMS-PCR. Direct sequencing of all samples was also performed to assess the accuracy of this method. DNA sequencing method validated the results of single tube H-ARMS-PCR.


RESULTS
We found full accordance for genotype adscription by sequencing method. The frequency of the AGT T521 and C702 alleles was significantly higher in CAD patients than in the control group (OR: 0.551, 95% CI: 0.359-0.846, P=0.008 and OR: 0.629, 95% CI: 0.422-0.936, P=0.028, respectively).


CONCLUSION
This is the first work describing a rapid, low-cost, high-throughput simultaneous detection of rs4762 and rs699 polymorphisms in AGT gene, used in large clinical studies.


Introduction
Several studies have established the evidence of a genetic linkage between the angiotensinogen gene (AGT) and essential hypertension (1,2). Angiotensinogen (AGT) is synthesized mainly by the liver and released into the circulation. More than 20 molecular variants have been identified in the AGT gene (3,4). Polymorphisms of particular interest are a C-to-T transition at nucleotide 521 (C521T) that causes the change of threonine 207 to methionine (T207M or rs4762) and a T to C base substitution at nucleotide 702 (T702C) that causes the change of methionine 268 to threonine (M268T or rs699). Both polymorphisms are located in exon 2 on chromosome 1 found to be strongly associated with essential hypertension and the pathogenesis of coronary disease (5)(6)(7). Different methods have been used to detect polymorphisms of AGT; among them are PCR amplification followed by restriction endonuclease digestion (8,9), PCR-Single-strand conformation polymorphism (PCR-SSCP) (10,11), real-time PCR (12,13) and Denaturing Gradient Gel Electrophoresis (DGGE) (14) further contributed to the discrepancy in the results. These techniques are relatively slow and very expensive in comparison to Hexa-primer-amplification refractory mutation system-PCR (H-ARMS-PCR). With this method, the genotyping could be done using only a thermocycler machine at the least time. In conventional ARMS PCR, the amplification of normal and mutant allele is done in two separate reactions, but in Multiplex ARMS-PCR, we can amplify both normal and mutant allele with a control fragment in a single reaction. The H-ARMS-PCR is performed by two non-allele-specific outer primers and four allele-specific inner primers in opposite orientation to each other. The outer primers amplify a large fragment of the target gene contains variant nucleotide as a control fragment and smaller allele-specific amplicons with different sizes that can easily be discriminated on gel electrophoresis either as homozygous or heterozygous. A deliberate mismatch at position -2 or -3 from the 3' terminal end of the inner primers can improve allele specificity (15).
Since rs4762 (T207M) and rs699 (M268T) polymorphisms are located in exon 2 of AGT gene and the distance between them is 182 bp, we developed a new H-ARMS-PCR for simultaneous detection of these SNPs in single tube.

Subjects and DNA isolation
In this case-control study, we recruited 148 CAD patient candidates for coronary artery bypass graft (CABG) surgery selected from 282 cases who referred to cardiac centers in Afshar Hospital (Yazd, Iran) due to symptoms of myocardial infarction from 2012 to 2015. Patients had major lesions (>50% narrowing of luminal diameter) in one, two, or three vessels (left anterior descending (LAD), left circumflex artery (LCX), and right coronary artery (RCA)) that were candidate for surgery. CAD patients were identified according to the coronary angiography guidelines (16). When up to 50% blockage was observed in the major epicardial coronaries and their branches associated with stenotic lesions, coronary arterial disease was considered present. The control group consisted of 135 unrelated healthy age, sex and ethnicity matched individuals with normal or near normal angiography reports (no lesion greater than 30%) ( Table 1). Yazd University Human Research Ethics Committee approved the study, in accordance with the revised declaration of Helsinki and all study participants gave their informed consents for the genetic analysis. Genomic DNA was extracted from peripheral blood samples using a salting out method.

Primer Design
Two common missense SNPs (T207M or rs4762and M268T or rs699) of AGT gene were selected (https://www.ncbi.nlm.nih.gov/snp/). The H-ARMS-PCR method for detection of two AGT polymorphisms was performed according to the tetra-primer PCR procedure (17). Six pri-mers were designed by the online website Primer 1:http://primer1.soton.ac.uk/primer1.html ( Table 2). The specificity of the primers was checked by 'BLAST' program at http://www.ncbi.nlm.nih.gov/blast. To enhance the specificity of inner primers, a destabilizing mismatch was incorporated at the third nucleotide from the 3'-terminus of each primer.

H-ARMS-PCR Analysis
PCR was performed in a total volume of 25 µL containing 50 ng of template DNA, 5 pmol of each outer primers, 10 pmol of each inner primers, 1X Multiplex PCR Master Mix (Yekta Tajhiz Azma Co., Tehran, Iran). PCR amplification (touchdown) was carried out at 95 ºC for 2 min, followed by denaturation at 95 ºC for 20 sec, first annealing at 70 ºC to 61 ºC (10 cycles). In the remaining cycles (25 cycles) annealing was carried out at 60 ºC for 1 min and extension at 72 ºC for 1 min, followed by a final extension for 5 min. Non-denaturation polyacrylamide gel electrophoresis (6%) and silver staining was used for separation and detection of PCR products. The rs4762 (C521T) and rs699 (T702C) polymorphisms are located in exon 2 AGT gene with 182 bp distance. Two primers, F1 and R1 were designed to amplify an 875 bp band, which served as control fragment for the success of amplification of two polymorphisms. Four spe-cific primers, F2 and R2 for rs4762 and F3 and R3 for rs699, with complementary 3'-termination to corresponding polymorphisms, were introduced (Table 2).
Here, we designate AGT gene with C and T nucleotide in position 521(C521T) as A and an allele respectively, and T and C nucleotide in position 702 (T702C) as B and b allele respectively. Therefore, four allele compositions are recognized for AGT gene including AB, Ab, aB and ab (schematic illustration in Fig. 1) and generated nine possible genotypes -AABB, AaBB, aaBB, aaBb, AABb, Aabb, AaBb, Aabb and aabb. The results were validated with direct DNA sequencing by using ABI 3700 capillary sequencer in 10% of samples.

Statistical Analysis
Unpaired Student's t-tests were used for comparison of distributions of continuous variables (as mean ± SD) in groups. The AGT gene SNPs was first assessed in three genotype categories (wildtype, heterozygote, and homozygote SNPs) and then grouped into two categories with heterozygotes and homozygote variants combined because of the dominant, the recessive, overdominant and log-additive models of inheritance observed for these polymorphisms. The association between two groups were examined by Chi-square goodness-of-fit test. Multiple logistic regression models (co-dominant, dominant and recessive) were employed to analyze the genetic data. Values of P<0.05 were regarded as statistically significant. The SPSS software (IBM SPSS 22, SPSS Inc., Chicago, IL, USA) was used for statistical analysis.

Results
The identification of each genotype (and Cis or Trans-state) was performed by comparing to the expected fragment lengths (Table 3). Table 3: Amplification patterns observed and those expected according to AGT polymorphism genotypes. A and a alleles are C and T nucleotides in position 521 (rs4762, C521T), respectively; B and b alleles are T and C nucleotides in position 702 (rs699, T702C), respectively; Cis is order of nucleotides as AB or ab in chromosome and Trans is order of nucleotides as Ab or aB in chromosome.  (Fig. 2). Ten percent of samples analyzed with H-ARMS-PCR were reevaluated by direct sequencing analysis. We obtained 100% accordance with both methods for genotype adscription. Coronary angiography revealed 148 patients (CAD + group) with one-vessel (LAD) (n= 35), two-vessels (LCX) (n= 59), or three-vessels (RCA) (n= 54) that were candidate for CABA (Coronary Artery Bypass Graft) and 135 patients (CADgroup) with no angiographically identified narrowing. Mean age (mean ± SD) was 53.9 ± 6.8 and 52.6 ± 6.9 yr for patients and controls, respectively. Genotype distributions and allelic frequencies of AGT polymorphisms among CAD patients and controls are shown in  (Table 3). Table 5 shows the results of the haplotype analysis. We found that AABb and aaBb haplotypes might significantly increase risk of CAD (P=0.029, OR: 0.460, 95%: 0.229-0.925, and OR: 3.146, 95% CI: 3.146-3.146, respectively).

Discussion
The AGT variants are considered to be a risk factor in various diseases and to be significant in body fluid hemostasis. Haplotype analysis showed a significant association between AGT rs4762 and rs699 with hypertension among Caucasian and Taiwan Chinese populations (18)(19)(20). The standard PCR instruments are highly desirable for scientific studies of large numbers of patients and for diagnostic analyses, economical and fast assays. Because these polymorphisms are risk factors for much morbidity, including cardiovascular disease and hypertension, here, we developed a rapid, sensitive and one-step hexa-primer PCR-ARMS method for detection rs4762 and rs699 polymorphisms. The most critical step for successful development of a new multiplex amplification refractory mutation system-PCR method is the primer design. In this study, the primers for amplification of AGT rs4762 and rs699 polymorphisms were designed using the online website Primer1: http://primer1.soton.ac.uk/primer1.html.  The primer sensitivity, specificity, and the annealing temperature were tested using separate tetraprimer ARMS-PCR for each SNP. In one reaction, T-ARMS-PCR assay was performed with F1, R1, F2 and R2 primers for detection of rs4762 polymorphism (Fig. 3, Line a) and in the other reaction, the T-ARMS-PCR assay was performed with F1, R1, F3 and R3 primers for detection of rs699 polymorphism (Fig. 3, Line b).
In addition, we obtained 100% accordance between direct DNA sequencing and H-ARMS-PCR for genotype adscription (Fig. 4). This assay is a reproducible and stable single-tube reaction.

Conclusion
We needed only a small amount of traditional PCR reagents and no special equipment. This procedure could be used by most clinical diagnostic laboratories. Additionally, this H-ARMS-PCR could be accomplished within 2.5 h following sample receipt, time-saving in the laboratory.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.